Motility activation and metabolism characteristics of spermatozoa of the black-lip-pearl oyster Pinctada margaritifera var: cumingii (Jameson, 1901)

Motility of Pinctada margaritifera (Linnaeus, 1758); var: cumingii (Jameson, 1901) (P. margaritifera) spermatozoa collected from gonads are not immediately activated at spawning in seawater (SW) but motility occurs when spermatozoa are transferred into alkaline seawater (pH ranging from 9.0 to 11.4). This motility-activating effect of alkaline pH is reversed when pH is shifted back to more acidic values. In both cases, activity of sperm (% motile cells) increases gradually after alkaline pH activation then lasts for several minutes. The characteristics of these fully motile spermatozoa are described in details at the level of flagella: the wave amplitude and wave-length range 5 to 6 μm and 15 μm respectively, while the flagellar beat frequency is approximately 49 Hz. The velocity of sperm displacement is from 220 to 230 μm/sec. The general swimming pattern is almost circular: the head trajectories describe portions of circles intercalated with small linear segments. Spermatozoa saved in natural seawater at 4°C retain potent motility for several days and can be subsequently activated by alkaline seawater. Respiration and ATP concentration were measured in 3 conditions: regular seawater (pH 7.8), artificial diluent (pH 8.2), and alkaline Tris-buffered seawater (pH 10.5). Results show that sperm respiration rates are higher whereas ATP levels are lower in the latter two media.     

Immunization against GnRH in the male camel (Camelus dromedarius): Effects on sexual behavior, testicular volume, semen characteristics and serum testosterone concentrations

The effect of immunization against gonadotrophin releasing hormone (GnRH) on sexual behavior, total scrotal size, semen characteristics and serum concentrations of testosterone, was evaluated for 24 wks in sexually mature camels (Camelus dromedarius). Eight bull camels were randomly divided into a treatment and control group. Four male camels were immunized using 2 mg GnRH - tandem-dimer conjugated to ovalbumin, (Pepscan Systems, the Netherlands) administered subcutaneously, 4 wks apart. Control male camels received the same amount of saline solution. Significant decline in serum testosterone level was observed in three immunized camels out of four, whereas one camel showed no effect. The testosterone levels reached to <1.0 ng/mL serum by week 4 after booster injection and remained suppressed through the course of the study. The total testicular volume was not affected until the end of the experiment. In treated animals, the sexual behavior negatively affected after the booster injection. Anti-GnRH vaccine had a seriously detrimental effect on the acrosin amidase activity and normal acrosome percentages in treated male camels. It is concluded that the vaccine was effective in reducing serum testosterone levels and libido, and it had a serious harmful effect on the acrosin amidase activity and percentages of spermatozoa with normal acrosome. The immunogen did not affect the total testicular volume.     

Use of amides as cryoprotectants in extenders for frozen sperm of tambaqui, Colossoma macropomum

Amides were tested as cryoprotectants in comparison with glycerol and DMSO (more traditional cryoprotectants) for recovery of Colossoma macropomum (tambaqui fish) sperm. Milt was extended in Beltsville Thawing Solution, then frozen with the addition of 2%, 5%, 8%, or 11% of: (1) dimethylacetamide (DMA), (2) dimethylformamide (DMF), (3) methylformamide (MF), or with 5% glycerol or 10% dimethylsulfoxide. Fertilization rates were greatest (P < 0.001) with amides; 8% DMF (91.6 ± 1.3%), 5% DMF (88.9 ± 1.6%), and 8% MF (83.0 ± 1.6%), which did not significantly differ among themselves, when compared with glycerol (51.6 ± 2.4%) and DMSO (61.9 ± 3.1%). The best hatching rates (P < 0.001) also occurred for 5% or 8% DMF and 8% MF (79.1 ± 3.1, 87.6 ± 1.5, and 74.8 ± 3.0, respectively) and were also similar (P > 0.05). For such treatments, both fertilization and hatching rates were similar (P > 0.05) to those with fresh sperm (91.7 ± 1.4 and 87.4 ± 1.4, respectively). The best sperm motility across extenders (at least 55.7%) was with 5%, 8%, and 11% DMF (P < 0.001). Those same treatments, along with 11% MF, provided the longest (P < 0.001) period of motility (at least 1 min). The greatest sperm integrity (more than 54%) was with 5% and 11% MF and with DMA and DMF at all tested concentrations (P < 0.001). The greatest (P < 0.001) sperm viability (at least 31%) was for 5%, 8%, and 11% DMA, and with 8% and 11% MF, and also for DMF at all tested concentrations. Sperm DNA integrity was best (more than 50%) for 2%, 5%, and 8% MF and for DMA and DMF at all concentrations (P < 0.001), whereas 2% DMA, 11% MF, 11% DMF, and the three amides at both 5% and 8% yielded the highest mitochondrial functionality (at least 44%; P < 0.001); thus, 8% MF and both 5% and 8% DMF were the cryoprotectants with the best postthaw quality for C. macropomum sperm.     

Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing

The objective of this study was to evaluate the effects of two commercially available semen extenders on the motility of cryopreserved goat sperm and to simplify the cryopreservation protocol. Individual goat ejaculates were split and processed in parallel for freezing in either commercially available soy-based extender (Bioxcell®) or egg yolk-based extender (Irvine TYB). Sperm quality was assessed using total and progressive sperm motility, measured by computer-assisted sperm analysis (CASA). Total motility was higher for samples processed in soy-based extender, both at pre-freeze (P = 0.002) and at post-thaw (P < 0.0001). Progressive motility was higher for semen processed in soy extender at post-thaw (P < 0.0001). Approximately 10% of samples processed in egg yolk-based extender had a large (> 50%) reduction in total motility prior to freezing. However, this type of extreme reduction in pre-freeze motility did not occur in semen samples processed in soy extender. In addition, the use of soy-based extender eliminated the need for a time-consuming sperm washing protocol. We concluded that a commercially available soy-based extender was superior to an egg yolk-based extender in preserving motility of cryopreserved goat sperm, using a two-step method.     

Maternal environment and the reproductive function of the offspring

Fetal programming of metabolic diseases is now a well established concept. The scope of the Developmental Origins of Health and Disease has, however, widened and led to the identification of new targets of fetal programming, notably effects on reproductive function. Epidemiologic studies about maternal nutrition and effects on offspring's fertility are rare, but a link between impaired fetal growth, possibly caused by maternal malnutrition, and reproductive function, has been established. The methodologic limitations inherent to human epidemiologic studies can be complemented through the use of animal models, which enable experimental studies on maternal environment and its effect on reproductive functions of the offspring. Altogether, an interaction between inappropriate maternal nutrition (excess or reduced nutritional intake, micronutrient unbalance, or alcohol intake) and reproductive maturation of the offspring has been shown in a majority of experiments as summarized in this review. The exact processes through which maternal nutrition or maternal environment affect reproductive function in the offspring remain unclear but epigenetic modifications are a clear link. Further studies are needed to better understand the mechanisms involved, identify the crucial critical periods, and prevent or treat the adverse effects.     

Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm

Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, semen was layered over continuous or discontinuous gradients. For Experiment 2, semen was layered over three column heights of continuous gradients in 15- or 50-ml conical-bottom tubes. For Experiment 3, increasing sperm numbers were layered over continuous gradient in 15- or 50-ml conical-bottom tubes. Following centrifugation, sperm pellets were evaluated for sperm morphologic quality, motility, DNA integrity, and recovery rate. Centrifugal fractionation improved (P < 0.05) sperm morphology, motility, and DNA integrity, as compared to controls. The continuous gradient increased (P < 0.05) sperm recovery rate relative to the discontinuous gradient, whereas sperm processed in 15-ml tubes yielded higher velocity and higher recovery rates (P < 0.05 for each) than that processed in 50-ml tubes. Sperm recovery rate was not affected (P > 0.05) by column height of gradient. Increasing sperm number subjected to gradient centrifugation decreased (P < 0.05) sperm recovery rate when 15-ml tubes were used.     

Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum)

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n = 42) obtained with the application of 11β-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX < 25%; II XX 25-50% and III XX > 50%. The average percentage of sperm motility was 18 ± 7% n = 12 (group I XX); 42 ± 6% n = 15 (group II XX) and 65 ± 12% n = 15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3 × 10⁶ spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.     

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen-thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.     

Automatic evaluation of ram sperm morphometry

This study was designed to develop a new method based on fluorescence microscopy and image analysis for the automatic assessment of sperm morphometry and to study separately the effect of drying and fixation on the parameters of head sperm morphometry in the ram. The study was divided into two experiments. In the first experiment, ejaculates from 25 adult males were collected using an artificial vagina, diluted and divided into four sample aliquots. The first was labeled directly with Hoechst 33342 (FRESH), and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying (DRIED), and the other were fixed either with glutaraldehyde (GLUT), or with methanol (MET), and labeled with Hoechst afterward. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and sperm heads were automatically captured and analyzed using the ImageJ program. The method used allowed a fast and automatic selection of most sperm heads for a given image with high precision. There was a general trend toward significant decrease in head length, width, area and perimeter of air-dried sperm compared with fresh sperm. On average, this decrease was of 4.1% in length, 4.3% in width, 9.1% in area, and 2.8% in perimeter. Between semen smears, fixation with glutaraldehyde significantly increased head sperm dimensions. The smears fixed with glutaraldehyde method is recommended for a more practical use than with fresh samples, providing better quality images than the other methods, and because the morphometric results obtained were more similar to the FRESH group than those of the DRIED and MET. In the second experiment, ejaculates from adult males were used to compare the sperm head morphometric results obtained with the new method developed (using the GLUT treatment as reference) with a more conventional CASMA method (semen smears stained with Hemacolor and processed with the ISAS commercial software, HEM). The GLUT method allowed the analysis of 100% of sperm, whereas only 93% of sperm could be analyzed using HEM. Spermatozoa displayed a bigger size when processed with HEM than with GLUT method in all primary sperm head morphometric parameters. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters. The new method developed allows automatic determination of sperm head morphometry in a reduced time, which facilitates its use in routine semen analysis. It was concluded that the automation of sperm morphometry is feasible using fluorescence microscopy and image analysis and that the effect of drying and fixation was less important than previously stated.